Mutational analysis of RsbT, an activator of the Bacillus subtilis stress response transcription factor, sigmaB

SigmaB, the stress-activated sigma factor of Bacillus subtilis, requires the RsbT protein as an essential positive regulator of its physical stress pathway. Stress triggers RsbT to both inactivate the principal negative regulator of the physical stress pathway (RsbS) by phosphorylation and activate a phosphatase (RsbU) required for sigmaB induction. Neither the regions of RsbT that are involved in responding to stress signaling nor those required for downstream events have been established. We used alanine scanning mutagenesis to examine the contributions of RsbT's charged amino acids to the protein's stability and activities. Eleven of eighteen rsbT mutations blocked sigmaB induction by stress. The carboxy terminus of RsbT proved to be particularly important for accumulation in Bacillus subtilis. Four of the five most carboxy-terminal mutations yielded rsbT alleles whose products were undetectable in B. subtilis extracts. Charged amino acids in the central region of RsbT were less critical, with four of the five substitutions in this region having no measurable effect on RsbT accumulation or activity. Only when the substitutions extended into a region of kinase homology was sigmaB induction affected. Six other RsbT variants, although present at levels adequate for activity, failed to activate sigmaB and displayed significant changes in their ability to interact with RsbT's normal binding partners in a yeast dihybrid assay. These changes either dramatically altered the proteins' tertiary structure without affecting their stability or defined regions of RsbT that are involved in multiple interactions.     

Expression of the ςB-Dependent General Stress Regulon Confers Multiple Stress Resistance in Bacillus subtilis

The alternative sigma factor sigmaB of Bacillus subtilis is required for the induction of approximately 100 genes after the imposition of a whole range of stresses and energy limitation. In this study, we investigated the impact of a null mutation in sigB on the stress and starvation survival of B. subtilis. sigB mutants which failed to induce the regulon following stress displayed an at least 50- to 100-fold decrease in survival of severe heat (54 degrees C) or ethanol (9%) shock, salt (10%) stress, and acid (pH 4.3) stress, as well as freezing and desiccation, compared to the wild type. Preloading cells with sigmaB-dependent general stress proteins prior to growth-inhibiting stress conferred considerable protection against heat and salt. Exhaustion of glucose or phosphate induced the sigmaB response, but surprisingly, sigmaB did not seem to be required for starvation survival. Starved wild-type cells exhibited about 10-fold greater resistance to salt stress than exponentially growing cells. The data argue that the expression of sigmaB-dependent genes provides nonsporulated B. subtilis cells with a nonspecific multiple stress resistance that may be relevant for stress survival in the natural ecosystem.     

Functional and structural characterization of RsbU, a stress signaling protein phosphatase 2C

RsbU is a positive regulator of the activity of sigmaB, the general stress-response sigma factor of Gram+ microorganisms. The N-terminal domain of this protein has no significant sequence homology with proteins of known function, whereas the C-terminal domain is similar to the catalytic domains of PP2C-type phosphatases. The phosphatase activity of RsbU is stimulated greatly during the response to stress by associating with a kinase, RsbT. This association leads to the induction of sigmaB activity. Here we present data on the activation process and demonstrate in vivo that truncations in the N-terminal region of RsbU are deleterious for the activation of RsbU. This conclusion is supported by comparisons of the phosphatase activities of full-length and a truncated form of RsbU in vitro. Our determination of the crystal structure of the N-terminal domain of RsbU from Bacillus subtilis reveals structural similarities to the regulatory domains from ubiquitous protein phosphatases and a conserved domain of sigma-factors, illuminating the activation processes of phosphatases and the evolution of "partner switching." Finally, the molecular basis of kinase recruitment by the RsbU phosphatase is discussed by comparing RsbU sequences from bacteria that either possess or lack RsbT.     

SOS-like induction in Bacillus subtilis: induction of the RecA protein analog and a damage-inducible operon by DNA damage in Rec+ and DNA repair-deficient strains.

We quantitated the induction of the Bacillus subtilis Rec protein (the analog of Escherichia coli RecA protein) and the B. subtilis din-22 operon (representative of a set of DNA damage-inducible operons in B. subtilis) following DNA damage in Rec+ and DNA repair-deficient strains. After exposure to mitomycin C or UV irradiation, each of four distinct rec (recA1, recB2, recE4, and recM13) mutations reduced to the same extent the rates of both Rec protein induction (determined by densitometric scanning of immunoblot transfers) and din-22 operon induction (determined by assaying beta-galactosidase activity in din-22::Tn917-lacZ fusion strains). The induction deficiencies in recA1 and recE4 strains were partially complemented by the E. coli RecA protein, which was expressed on a plasmid in B. subtilis; the E. coli RecA protein had no effect on either induction event in Rec+, recB2, or recM13 strains. These results suggest that (i) the expression of both the B. subtilis Rec protein and the din-22 operon share a common regulatory component, (ii) the recA1 and recE4 mutations affect the regulation and/or activity of the B. subtilis Rec protein, and (iii) an SOS regulatory system like the E. coli system is highly conserved in B. subtilis. We also showed that the basal level of B. subtilis Rec protein is about 4,500 molecules per cell and that maximum induction by DNA damage causes an approximately fivefold increase in the rate of Rec protein accumulation.     

The yeast two-hybrid system detects interactions between Bacillus subtilis sigmaB regulators.

SigmaB, the general stress response sigma factor of Bacillus subtilis, is regulated by the products of seven genes (rsbR, S, T, U, V, W, and X) with which it is cotranscribed. Biochemical techniques previously revealed physical associations among RsbW, RsbV, and sigmaB but failed to detect interactions of RsbR, S, T, U, or X with each other or RsbV, RsbW, or sigmaB. Using the yeast two-hybrid system, we have now obtained evidence for such interactions. The yeast reporter system was activated when RsbS was paired with either RsbR or RsbT, RsbR was paired with RsbT, and RsbV was paired with either RsbU or RsbW. In addition, RsbW2 and RsbR2 dimer formation was detected. RsbX failed to show interactions with itself or any of the other sigB operon products.     

Stress activation of Bacillus subtilis sigma B can occur in the absence of the sigma B negative regulator RsbX.

Environmental stress activates sigma B, the general stress response sigma factor of Bacillus subtilis, by a pathway that is negatively controlled by the RsbX protein. To determine whether stress activation of sigma B occurs by a direct effect of stress on RsbX, we constructed B. subtilis strains which synthesized various amounts of RsbX or lacked RsbX entirely and subjected these strains to ethanol stress. Based on the induction of a sigma B-dependent promoter, stress activation of sigma B can occur in the absence of RsbX. Higher levels of RsbX failed to detectably influence stress induction, but reduced levels of RsbX resulted in greater and longer-lived sigma B activation. The data suggest that RsbX is not a direct participant in the sigma B stress induction process but rather serves as a device to limit the magnitude of the stress response.     

Phosphorylation and RsbX-dependent dephosphorylation of RsbR in the RsbR-RsbS complex of Bacillus subtilis

In the pathway that controls sigmaB activity, the RsbR-RsbS complex plays an important role by trapping RsbT, a positive regulator of sigmaB of Bacillus subtilis. We have proposed that at the onset of stress, RsbR becomes phosphorylated, resulting in an enhanced activity of RsbT towards RsbS. RsbT is then free to interact with and activate RsbU, which in turn ultimately activates sigmaB. In this study with purified proteins, we used mutant RsbR proteins to analyze the role of its phosphorylatable threonine residues. The results show that the phosphorylation of either of the two RsbT-phosphorylatable threonine residues (T171 and T205) in RsbR enhanced the kinase activity of RsbT towards RsbS. However, it appeared that RsbT preferentially phosphorylates T171. We also present in vitro evidence that identifies RsbX as a potential phosphatase for RsbR T205.     

Purification of an SOS repressor from Bacillus subtilis.

We have identified in Bacillus subtilis a DNA-binding protein that is functionally analogous to the Escherichia coli LexA protein. We show that the 23-kDa B. subtilis protein binds specifically to the consensus sequence 5'-GAACN4GTTC-3' located within the putative promoter regions of four distinct B. subtilis DNA damage-inducible genes: dinA, dinB, dinC, and recA. In RecA+ strains, the protein's specific DNA binding activity was abolished following treatment with mitomycin C; the decrease in DNA binding activity after DNA damage had a half-life of about 5 min and was followed by an increase in SOS gene expression. There was no detectable decrease in DNA binding activity in B. subtilis strains deficient in RecA (recA1, recA4) or otherwise deficient in SOS induction (recM13) following mitomycin C treatment. The addition of purified B. subtilis RecA protein, activated by single-stranded DNA and dATP, abolished the specific DNA binding activity in crude extracts of RecA+ strains and strains deficient in SOS induction. We purified the B. subtilis DNA-binding protein more than 4,000-fold, using an affinity resin in which a 199-bp DNA fragment containing the dinC promoter region was coupled to cellulose. We show that B. subtilis RecA inactivates the DNA binding activity of the purified B. subtilis protein in a reaction that requires single-stranded DNA and nucleoside triphosphate. By analogy with E. coli, our results indicate that the DNA-binding protein is the repressor of the B. subtilis SOS DNA repair system.